The denatured state of N-PGK is compact and predominantly disordered.
The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I(b)) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I(k)) and denatured (D)]. The transition to I(b) is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I(b). Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I(b) were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I(b) indicate that it forms from the fully denatured state within 100 micros, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I(b) is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.
Cliff
Matthew J
MJ
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK.
Craven
C Jeremy
CJ
Marston
James P
JP
Hounslow
Andrea M
AM
Clarke
Anthony R
AR
Waltho
Jonathan P
JP
D001407:Bacillus / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D017433:Protein Structure, Secondary; D013113:Spin Labels; D013816:Thermodynamics
D013113:Spin Labels; D010735:Phosphoglycerate Kinase; D019791:Guanidine